Cell membrane proteins represent the targets of more than 60% of pharmaceuticals currently on the market, including asthma medications (e.g. inhaled albuterol which targets membrane “fight-or-flight” receptors), cancer therapies (e.g. Herceptin which targets membrane growth factor receptors), and dermatologic agents (e.g. Raptiva which targets membrane cell adhesion molecules). To identify drugs and increase the likelihood of eventual efficacy, there has been intense interest in the development of high throughput, high information-content assays for screening drug candidates against membrane proteins in the context of their native cell membrane environment. Traditional methods have relied on expressing target proteins in live cells and performing binding assays with libraries of drug candidates. While elegant in theory, these live cell assays are somewhat labor-intensive and require expertise in cell culture. Moreover, cell-to-cell variation in both target protein expression and binding efficiency of drug candidates makes these assays essentially semi-quantitative. Variation in the size and shape of cells also makes them somewhat problematic for certain instrument detection systems.
The inherent complexity of live cell assays has stimulated the development of technology for performing high throughput, high information-content assays of cell membrane activity under in vitro conditions. A number of technologies have focused on ways of displaying cell membranes in an in vitro environment under conditions which preserve the function of cell membrane components. The MembraneChip™, developed by Synamem Corporation (U.S. Pat. No. 6,699,719; U.S. Pat. No. 6,228,326; Groves et al. (1997) Science, 275, 651-653) is a surface detector device for arraying phospholipid membranes that consists of planar lipid bilayer membranes (either synthetic or native cell membranes) arrayed on fused silica, which is particularly useful for high throughput parallel assays including ligand/receptor interactions and live cell-cell signal transduction (Yamazaki et al. (2005) BMC Biotechnol., 5:18). This technology is predicated upon the observation that small unilamellar vesicles (SUVs), liposomes having diameters ranging from 25 nm to 5 μm, fuse with glass or silica in a manner which generates planar lipid bilayers (Brian and McConnell (1984) Proc. Natl. Acad. Sci. U.S.A., 81:6159-6163; Groves and Boxer (2002) Acc. Chem. Res., 35:149-157). Arrayed membranes retain a physiological level of fluidity characteristic of the in vivo environment, a property critical for numerous receptor interactions and signal transduction pathways including G protein-coupled receptor (GPCR), receptor tyrosine kinase, and T cell receptor signaling.
In a manner analogous to displaying supported lipid bilayers on planar substrates, it is also possible to deposit a single lipid bilayer (e.g. comprising dimyristoyl phosphatidylcholine (DMPC)) on commercially-available glass or silica beads (1-50 μm in diameter) (Bayer) and Bloom, (1990) Biophys. J., 58:357-362). As in the case of the MembraneChip™, these membrane-derivatized beads display physiologically fluid lipid bilayers. Membranes are typically separated from the silica bead by a thin film of water as they are with planar substrates, facilitating the long-range lateral mobility of lipids and bilayer components. Membrane-derivatized beads have great utility for measuring processes ranging from ligand/receptor binding to complex cell-cell interactions. Moreover, the phenomenon of pair-wise interactions among beads in a batch has been leveraged to generate label-free assays of ligand/receptor interactions (Baksh et al. (2004) Nature, 427:139-141). Indeed, cholera toxin B binding to ganglioside GM1 displayed in membrane-derivatized beads has been detected using the statistical analysis of colloidal pair-wise distribution (Baksh et al. 2004).
There have been a number of studies using beads for isolating cell membranes for purposes of purification (Jacobson and Branton, (1977) Science, 195:302-304; Cohen et al. (1977) J. Cell Biol., 75:119-134; Kinoshita et al. (1979) J. Cell Biol., 82:688-696). However, these approaches invariably lead to beads coated with native cell membranes which are in an orientation with the extracellular leaflet closest to the bead surface and the intracellular leaflet exposed to the bulk solution, rendering potential membrane components on the external leaflet inaccessible to various probes. Given that quite a number of test agents and drug candidates interact with the extracellular domains of cell membrane proteins, this limitation makes it particularly challenging to use these earlier methods for investigating cell surface proteins, as well as other cell membrane components. Indeed, more recent results using erythrocyte membranes on silica beads demonstrated similar results (Kauffmann and Tanaka, (2003) Chemphyschem, 4:699-704), suggesting the difficulty in obtaining beads coated with membranes capable of displaying the external leaflet of the plasma membrane. These earlier methods thus generate membrane-coated particles in which the internal side of the cellular membrane is exposed to the bulk phase as opposed to the more desirable orientation whereby the external side of the membrane is exposed. Consequently, the present methods and compositions enable a considerably more useful orientation of the cell surface on a bead particle, as well as a more practical solution for in vitro analysis of cell membrane components.
Given the vast market opportunity for drugs targeting cell membrane components, there remains a tremendous need for developing technology for displaying native cell membranes in such a manner as to mimic the natural orientation of cellular membrane components on a variety of supports to enable rapid, high throughput, high information-content assays.